This proposal is for support of research to study the mechanism involved in the initiation of DNA replication in eukaryotes. The autonomously replicators in yeast, are strong candidates for origins of replication.. These ARS elements contain an 11 base-pair consensus sequence and two domains of unusual DNA structures that are required for normal replication patterns in yeast cells. A major goal of the studies described in this application is to construct an artificial ARS that contains the structural features that are highly conserved in the natural ARS elements. The artificial ARS will be produced from a series of synthetic oligonucleotides that possess altered DNA structures. It will then be shown that the artificial ARS function as a replication when introduced into yeast and human cells and the role of each ARS structure in the control of replication will be determined. INformation gained from these studies will be used to refine the model for ARS structure which will, in turn, be used to identify functional ARS elements in the yeast genome. The cell-cycle regulation of the artificial ARS will also be examined in order to identify the point in the cell cycle at which a given structure exerts its effects on the replication process. Finally, the chromatin packaging and protein binding activity of the artificial ARS will be studied in order to assess the role of unusual DNA structures in the control of protein-DNA interactions. These studies, if successful, will enable us to determine the role of each ARS structure in the control of origin function and will provide a better understanding of DNA replication in eukaryotes.